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Objective:To investigate the effect of interleukin (IL)-23 receptor (IL-23R) overexpression on the balance of T helper 17 (Th17 cells)/regulatory T cells (Treg cells) in experimental autoimmune uveitis (EAU) mice.Methods:Twelve 8-week-old female C57BL/6J mice were randomly divided into LV-Ctrl group and LV-IL-23R group, with 6 mice in each group. Two groups of mice were injected with LV-Ctrl and LV-IL-23R lentiviruses through the tail vein, respectively; 7 days after injection, the EAU mouse model was established by active immunization with vitamin A-binding protein 1-20 between photoreceptors. Starting from 13 days after immunization, the fundus of the mice was observed by indirect ophthalmoscopy every 2 days and clinical scores were performed; 30 days after immunization, hematoxylin-eosin staining was used to observe the histopathological changes of mouse retina. The levels of IL-17 in serum of the two groups of mice were detected by enzyme-linked immunosorbent assay; the proportion of Th17 cells and Treg cells was detected by flow cytometry. The relative mRNA expression of IL-23R, IL-17, retinoic acid-related orphan receptor γt (RORγt), IL-10 and forkhead transcripyion factor p3 (Foxp3) were detected by real-time quantitative polymerase chain reaction. Comparisons between groups were performed using repeated measures analysis of variance, independent samples Mann-Whitney U test, and independent samples t test. Results:Compared with the LV-Ctrl group, the retinal inflammatory reaction of the LV-IL-23R group was more severe. At 13 days after immunization, there was no significant difference in fundus inflammation scores between LV-IL-23R group and LV-Ctrl group ( t=-2.001, P=0.058); 15-29 days after immunization. The fundus inflammation scores of LV-IL-23R group were higher than those of LV-Ctrl group, and the difference was statistically significant ( t=-4.429,-6.578, -7.768, -10.183, -6.325, -7.304, -4.841, -6.872; P<0.001). Histopathological examination showed that the infiltration of inflammatory cells in the fundus increased, the retinal structure was damaged more seriously, and the histopathological score was significantly increased, and the difference was statistically significant ( t=-4.339, P=0.001). Compared with the LV-Ctrl group, the relative expression of IL-23R mRNA in the spleen of the LV-IL-23R group was significantly increased, and the difference was statistically significant ( Z=2.087, P=0.037). The relative expression of IL-17 and RORγt mRNA increased, while the relative expression of IL-10 and Foxp3 mRNA decreased, and the differences were statistically significant ( t=-6.313,-5.922, 4.844, 7.572; P=0.003, 0.004, 0.008, 0.002). Compared with the LV-Ctrl group, the level of IL-17 in the serum of the mice in the LV-IL-23R group was significantly increased, and the difference was statistically significant ( t=-5.423, P=0.002); the proportion of Th17 cells in the spleen and lymph nodes was significantly increased, whereas, the proportion of Treg cells was significantly reduced, and the difference was statistically significant ( t=-4.290, 3.700; P=0.002, 0.006). Conclusion:IL-23R overexpression can promote Th17/Treg imbalance in EAU mice, and aggravate the clinical and pathological manifestations of EAU.
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Objective@#To investigate the effect of human umbilical cord mesenchymal stem cells derived exosomes (hUC-MSC-exo) on the phenotype of peripheral blood macrophages from rabbit autoimmune dry eye and the expression of related cytokines.@*Methods@#The hUC-MSCs were isolated and characterized.Exosomes derived from hUC-MSCs were extracted by ultracentrifugation and observed directly using electronic microscopy.Specific markers of exosomes were analyzed by Western blot.Six rabbits were randomly divided into the normal control group and the dry eye group by using the random number table method, 3 rabbits for each group.Rabbit model of autoimmune dry eye was established in the dry eye group, and the lacrimal glands were collected for quantitative real-time PCR (qRT-PCR) at the 8th week.In vitro, activated peripheral blood mononuclear cells (PBMCs) from rabbit autoimmune dry eye model were incubated with hUC-MSC-exo or phosphate buffered saline (PBS). After 48 hours, cells from the hUC-MSC-exo group and the PBS control group were collected.The mRNA expression levels of related cytokine genes and subpopulation-related marker genes in macrophages were quantified by qRT-PCR.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.This study protocol was approved by Ethic Committee of Tianjin Medical University Eye Hospital (No.TJYY20181217001). Written informed consent was obtained from each family before obtaining umbilical cord.@*Results@#Exosomes derived from hUC-MSCs had typical morphology and specific markers.qRT-PCR results showed that, the relative expression quantity of M1 macrophages phenotypic molecular nitric oxide synthase 2 (NOS2) mRNA and inflammatory factor tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) mRNA in the lachrymal organization in the dry eye model group was significantly higher than those in the normal control group (3.06±1.00 vs. 1.00±0.03, 2.77±0.72 vs. 1.01±0.02 and 1.30±0.08 vs. 1.01±0.01, respectively), the relative expression quantity of M2 macrophages phenotypic molecular arginase 1 (Arg1), CD206 and IL-10 mRNA in the lachrymal organization in the dry eye model group was significantly lower than those in the normal control group (0.55±0.07 vs. 1.00±0.00, 0.60±0.13 vs.1.00±0.00, 0.65±0.14 vs. 1.01±0.01, respectively), with significant differences between them (all at P<0.05). In vitro, the relative expression quantity of M1 macrophages phenotypic molecular NOS2 mRNA and inflammatory factor TNF-α, IL-1β mRNA in PBMCs in the hUC-MSC-exo group was significantly lower than those in the PBS control group (0.59±0.08 vs.0.98±0.03, 0.56±0.07 vs. 1.03±0.11, 0.47±0.04 vs.1.00±0.08)(all at P<0.05); the relative expression quantity of M2 macrophages phenotypic molecular Arg1, CD206 mRNA and anti-inflammatory cytokine TGF-β, IL-10 and IL-4 mRNA in PBMCs in the hUC-MSC-exo group was significantly higher than those in the PBS control group (2.13±0.28 vs. 1.10±0.17, 1.32±0.03 vs. 1.01±0.06, 1.53±0.20 vs. 1.05±0.10, 1.47±0.08 vs.0.98±0.03, 1.51±0.16 vs. 1.01±0.03), with significant differences between them (all at P<0.05).@*Conclusions@#hUC-MSC-exo can polarize peripheral blood macrophages toward immune-suppressive M2-like phenotype, inhibit the production of pro-inflammatory cytokines TNF-α and IL-1β, and meanwhile increase the expression of anti-inflammatory factors IL-10 and TGF-β.
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The effect of macrophage on IL-2 production by tonsillar T cells was investigated. The human tonsillar mononuclear cells were purified by a two-step procedure including depletion of plastic-adherent cel1s and rosetting with SRBC. The purity of Enriched T cel1s (ETC) was 98%. It was found that IL-2 production was greatly decreased in ETC by removing adherent cells and could be completely restored by replacing the adherent cells. The IL-2 production in the same extent was induced in ETC when TPA was substituted for adherent cells and the synergistic effect of TPA and macrophages greatly augmented the IL-2 secretion by ETC. Incubation of ETC with high concentration of PHA or ConA alone resultes in efficient IL-2 production. These results indicated that IL-2 production was macrophage dependent.